Our understanding of efferocytosis or how the cell corpses are removed has been largely impeded by lack of amenable systems to study this process. This problem has been particularly intractable from the perspective of the dying cell. We recently developed a pH-based readout of efferocytosis that allows for reliable visualization and quantitative measurements of images from Drosophila cell culture and in vivo development. Our novel cell- based assay enables high-throughput analysis to identify genes necessary to produce eat-me signals in apoptotic cells. In addition to pursuing a candidate gene-approach, we will implement high-throughput RNAi in Drosophila cells. By integrating several powerful tools including the whole-genome RNAi libraries, high- throughput high-content imaging, computational image analysis, the extensive preexisting genetic RNAi toolkit in flies, classical Drosophila genetics, and a vast amount of pre-compiled gene expression data, we will identify novel genes involved in efferocytosis. Additional assays will help us place the identified genes into broad categories such as pro-apoptotic genes or genes required for phosphatidylserine exposure. To assess the function of gene candidates in vivo, we will measure the corpse load in the macrophages of RNAi-injected Drosophila embryos. An analysis in the parallel process of the removal of injured neurons by glia in adult flies with genetically driven RNAi in neurons will enable us to identify the most broadly-functional signals. The proposed research will lead to identification of genes that promote efferocytosis and will form the basis of our long-term studies in flies and vertebrate systems to better understand efferocytosis and apoptosis in human diseases and to modulate the underlying pathways. The knowledge gained in our studies could be applied to increase the eat-me signaling in diseases where efferocytic activity is diminished, such as cancer, atherosclerosis, a number of respiratory diseases, and autoimmune disorders that include systemic lupus erythematous and rheumatoid arthritis. Our understanding of efferocytosis is also important for disorders that include neurodegeneration, where this process may be up-regulated.